Living Image 3.1

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8. Spectral Unmixing 8.1 Spectral Unmixing 114 1. Load the image sequence. In this example, the fluorophore is Quantum Dots 800. Images were acquired using a 675 nm excitation filter and emission filters from 720 to 820 nm in 20 nm increments. 2. In the Analyze tab of the Spectral Unmixing tools, put a check mark next to the emission wavelengths that you want to include in the analysis. Excitation wavelength Emission wavelengths of the sequence 3. In the Components drop-down list, select the number of spectral components to unmix in the images (the number of fluorophores +1 since tissue autofluorescence accounts for 1 component). For example, if the image data includes one fluorophore, then there are two components to unmix−the fluorophore signal and the tissue autofluorescence.
Living Image ® Software User’s Manual 4. Make a selection from the Mask drop-down list: Photo - Opens the Photo Mask Setup window. The purple mask specifies the area for analysis. a. Use the Threshold slider or arrows to adjust the mask so that it matches the underlying subject photograph as closely as possible without including any area outside the subject image. b. Click Set. All - The entire image is analyzed. This option is not recommended. It introduces many offtarget pixels that might mislead and significantly slow down the analysis ROI - ROIs applied to the sequence are available in the drop-down list. Select an ROI to analyze just the area in the ROI. This option is recommended for well plates or dark objects where it is hard to achieve a good mask from the photograph. 5. In the tool palette, click Unmix Spectra. - The spectral unmixing results appear. The Distribution tab shows a photon density map of each unmixed result and a composite image that includes all of the fluorescent signals, each displayed in a different color. Unmixed 1 = Autofluorescence Composite = Unmixed 1 + Unmixed 2 Unmixed 2 = Fluorophore signal 115
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Living Image 3.1

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